Article Figures & Data
Figures
- Figure 1. HIPK2 regulates spastin at posttrascriptional levels.
(A) Indicated cells were transfected with a mix of three human HIPK2-specific (siHIPK2) or negative control (siCtr) stealth siRNAs and analysed by Western blot (WB) 96 h posttransfection. Unless otherwise indicated, here and in the following figures representative WB of three independent experiments was shown and the intensity of the spastin bands quantified, normalized with GAPDH, and reported in a.u. relative to control, as mean ± SD of three independent experiments. Molecular weight markers are reported in kilodalton. Here, spG311/1 Ab was used for spastin detection and statistical differences are relative to corresponding siCtr, unpaired t test. (B) HeLa cells were transfected with vectors expressing control LacZ sh-RNA (sh-Ctr) or a mix of two HIPK2-specific shRNAs (sh-HIPK2) and analysed by WB 72 h posttransfection (left panels). HeLa HIPK2-Cas9 cells and their parental control cells (Ctr-Cas9) were analysed by WB 24 h after plating (right panels). Statistical differences are relative to corresponding control, unpaired t test. (C) HeLa HIPK2-Cas9 cells were transfected with low dose of HIPK2-HA–expressing vector to avoid apotosis induction and analysed by WB 24 h posttransfection with anti-HIPK2 and anti-spastin Abs. Statistical difference is relative to empty vector transfected cells used as control, unpaired t test. (D) NSC34 cells were transfected with a mix of three mouse HIPK2-specific (siHIPK2) or negative control (siCtr) stealth siRNAs and incubated in differentiation medium; 5 d post siRNA transfection cells were analysed by WB. Statistical difference, unpaired t test. (E) Cortical neurons at 5 days in vitro (DIV5) derived from indicated mouse infected with adenovirus expressing the recombinase CRE were analysed by WB 96 h postinfection. Statistical difference, unpaired t test. (F, G) Indicated neural tissues were explanted from Hipk2+/+ and Hipk2KOF/KOF adult mice (5 mo) and spastin levels were analysed by WB and real time RT-PCR. (F) Representative WB showing the reduction of spastin isoforms is reported; statistical differences are relative to corresponding control tissue, unpaired t test. (G) Relative fold-change of spastin mRNA levels, using actin mRNA as normalizer, are represented as mean ± SD of three independent experiments in (G); ns, no statistically significance, unpaired t test. (H) Representative WB of HeLa cells transfected with GFP (empty) or GFP-HIPK2–expressing vectors and lysed 24 h posttransfection. Statistical difference, unpaired t test. (I, J) HeLa cells transfected as in Fig 1A were analysed by WB and real-time RT-PCR at the indicated time post transfection. In (I), representative WB, statistical differences are relative to corresponding siCtr, unpaired t test; in (J), relative fold-change of spastin mRNA levels, using GAPDH mRNA as normalizer, as mean ± SD of three independent experiments. ns, unpaired t test.
Source data are available for this figure.
Source Data for Figure 1[LSA-2020-00799_SdataF1.1.xlsx][LSA-2020-00799_SdataF1.2.tif]
- Figure S1. Spastin expression is regulated by HIPK2.
(A) Total cell extracts of A549 cells shown in Fig 1A were run twice and Western blot (WB) analysis performed by using the sp6C6 monoclonal spastin Ab for spastin detection. Representative WB and its quantification are reported. Statistical difference, unpaired t test. (B, C) NSC34 cells were incubated in differentiation medium. Differentiation was evaluated 5 d after treatment by IF and WB using β3-tubulin neuronal marker. In (B), representative images of β3-tubulin staining at indicated days after differentiation are reported. Scale bar, 10 μM. In (C), representative WB with indicated Abs is shown. (D) Indicated cortical neurons were analysed by PCR using primers amplifying cKO and KO Hipk2 alleles. The effect of CRE recombinase in Hipk2cKO/cKO is showed by the amplification of the KO allele. (E) PCR genotyping analysis of the genomic DNA extracted from tails derived from indicated mice by using primers specifically amplifying WT (P-WT) or KOF Hipk2 alleles (P-KOF). (F) HIPK2 mRNA residual levels were analysed by real time RT-PCR in tissues analysed in Fig 1F and G. Relative fold-change of HIPK2 mRNA levels, using actin mRNA as normalizer, are represented as mean ± SD of three independents. Statistical differences, relative to +/+ counterparts, unpaired t test. (G) HeLa cells were incubated in standard culture condition at 37°C (mock) or shifted to 42°C for 1 h to induce HIPK2 stabilization and analysed by WB with indicated Abs. Statistical difference, unpaired t test. (H) HeLa cells transfected as in Fig 1A were analysed by real-time RT-PCR at the indicated time post transfection. Relative fold-change of HIPK2 mRNA levels, using GAPDH mRNA as normalizer, are represented as mean ± SD of three independent experiments. Statistical differences, unpaired t test.
- Figure 2. HIPK2 regulates spastin via proteasomal degradation through K554 polyubiquitination.
(A) Representative Western blot (WB) of HeLa cells transfected as in Fig 1A and lysed 96 h posttransfection and 8 h after treatment with 20 μM MG132 or its solvent DMSO. MDM2 stabilization has shown as MG132 positive control. Statistical differences, ANOVA test. (B) HeLa cells were transfected as in Fig 1A and harvested 96 h posttransfection and 8 h after treatment with 20 μM MG132 or DMSO. Total cell extract (TCE) was analysed by WB for the indicated proteins and immunoprecipitated with anti-spastin Ab. IPs were analysed by WB with anti-Ub and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a nonspecific band. The intensity of spastin-Ub ladder was normalized by the intensity of spastin band in IP and reported relative to siCtr DMSO–treated cells as mean ± SD (n = 3). Statistical differences, ANOVA test. (C) NSC34 cells were transfected as in Fig 1B and harvested 5 d posttransfection and 8 h after treatment with 20 μM MG132. TCE was analysed by WB for the indicated proteins and immunoprecipitated with anti-spastin Ab. IPs were analysed by WB with anti-Ub and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a specific band. The intensity of spastin-Ub ladder was calculated and reported as in Fig 2B. Statistical differences, ANOVA test. (D) HeLa Ctr-Cas9 and HIPK2-Cas9 cells were transfected with vectors expressing HA-tagged Ub-WT (Ub-HA) or its derivative KoUb-HA (i.e., Ub with all lysines mutated in arginines) and treated 24 h posttransfection with 20 μM MG132 or DMSO for 8 h. TCEs were analysed as in Fig 2B. IPs were analysed by WB with anti-HA and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a nonspecific band. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin band in IP and reported relative to the correspondent DMSO-treated cells as mean ± SD (n = 3). Statistical differences, ANOVA test. (E) Spastin amino acid sequence encompassing the K554 is reported for indicated organisms. Fly = Drosophila melanogaster; worm = Caenorhabditis elegans. (F) HIPK2-Cas9 HeLa cells were transfected with vectors expressing flag-myc–tagged spastin-WT or spastin-K554R in combination with the vector expressing HA-tagged Ub-WT and treated 24 h posttransfection with 20 μM MG132 for 8 h. TCE were analysed by WB and immunoprecipitated with anti-Flag Ab (mouse Ab by Origene Technologies). IPs were analysed by WB with anti-HA and anti-Flag Ab (rabbit Ab by Sigma-Aldrich). The arrows indicate the position of the unmodified spastin isoforms. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin bands in IP and reported as mean ± SD (n = 4). Statistical difference, unpaired t test. (G) HIPK2-Cas9 and Ctr-Cas9 HeLa cells were transfected with vectors expressing spastin-WT or spastin-K554R in combination with peGFP vector at 10:1 molar ratio and analysed by WB 24 h posttransfection. GFP expression was used as internal control for transfection efficiency. Representative WB is shown. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to correspondent Ctr-Cas9 control cells. Statistical differences, ANOVA test.
Source data are available for this figure.
Source Data for Figure 2[LSA-2020-00799_SdataF2.1.xlsx][LSA-2020-00799_SdataF2.2.tif]
- Figure S2. Spastin is K48-polyubiquitinated.
(A) Data quantification of spastin/loading control levels by Western blot (WB) on total cell extract (TCE), relative to data shown in Fig 2B. Quantification was performed as in Fig 2A. Bars are mean ± SD of three independent experiments; Statistical differences, ANOVA test. (B) As in Fig 2D, HeLa cells were transfected with vector expressing Ub-HA and treated 24 h posttransfection with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-spastin mouse monocolonal Ab, sp3G11/1. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. (C, D) HeLa HIPK2-Cas9 and Ctr-Cas9 cells were transfected with vectors expressing HA-Ub or its derivative K48-Ub-HA (i.e., Ub with only K48, the other lysines are mutated in arginines) and treated 24 h post transfection with 20 μM MG132 or DMSO for 8 h. TCE were analysed by WB and IP as in Fig 2D. In (C), representative WB of three independent experiment is shown. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. In (D), relative spastin-Ub-HA levels were quantified and reported as in Fig 2C. Statistical differences, unpaired t test. (E) As in Fig 2F, HeLa cells were transfected with vectors expressing flag-myc tagged spastin in combination with Ub-HA and 24 h post transfection cells were treated with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-Flag mouse monoclonal Ab. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-Flag Abs. The arrow indicates the position of the unmodified spastin.
- Figure 3. Kinase activity of HIPK2 regulates spastin protein levels.
(A) Representative Western blot (WB) of HeLa (right panels) and NSC34 cells (left panels) transfected with vectors expressing HA-tagged HIPK2-WT, its derivative -K228A mutant or HA-alone (empty). Statistical differences, Anova test. (B) Representative WB of HeLa cells transfected with vectors expressing flag-myc spastin-WT or indicated spastin mutants in combination with peGFP vector at 10:1 molar ratio and lysed at indicated time post transfection. GFP expression was used as internal control for transfection efficiency. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to spastin-WT for each time point. Statistical differences, ANOVA test. (C) HeLa cells were transfected with vectors expressing flag-myc–tagged spastin-S268A or spastin-WT, treated with 25 μg/ml cycloeximide 36 h posttransfection and analysed by WB at indicated times after treatment. Note that to minimize differences in spastin levels at the time 0, cells were transfected with different doses of the expressing vectors, that is, 1 μg of spastin-S268A–expressing vector and 0.5 μg of spastin-WT–expressing vectors. Representative WB is shown. The levels of spastin-Flag bands relative to those of GAPDH were measured at each time point and reported as mean ± SEM of four different independent experiments in the right panel. Statistical differences were calculated and reported for each time point, unpaired t test.
Source data are available for this figure.
Source Data for Figure 3[LSA-2020-00799_SdataF3.1.xlsx][LSA-2020-00799_SdataF3.2.tif]
- Figure 4. Phosphorylation in S268 prevents spastin polyubiquitination and degradation.
(A, B) HeLa Ctr-Cas9 (A) and HIPK2-Cas9 (B) cells were transfected with vectors expressing indicated flag-myc–tagged spastin-WT or its derivative mutants in combination with the vector expressing HA-tagged Ub-WT and treated 24 h posttransfection with 20 μM MG132 for 8 h. Total cell extracts (TCEs) were analysed by Western blot (WB) and IP as in Fig 2F. Samples were processed in parallel and analysed on the same blot to make comparison between HIPK2 proficient and null cells. The arrows indicate the position of unmodified spastin isoforms. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin-Flag bands in IP and reported as mean ± SD (n = 3). Statistical differences, ANOVA test. (C) HeLa Crt-Cas9 and HIPK2-Cas9 cells were transfected with vectors expressing indicated flag-myc–tagged spastin WT or its derivative S268D in combination with peGFP vector as in Fig 2G and analysed by WB 24 h post transfection. GFP expression was used as internal control for transfection efficiency. Representative WB is shown. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to Ctr-Cas9 control cells. Statistical differences, Anova test. (D) Representative Co-IP showing that spastin-S268D preferentially binds CAND1. HeLa cells were co-transfected with the plasmid expressing MYC-CAND1 in combination with empty vector or vectors expressing spastin-S268A or spastin-S268D. Cells were collected 24 h posttransfection. TCE were analysed by WB or immunoprecipitated with anti-Flag or anti-CAND1 Abs and analysed as indicated. The asterisk indicates an aspecific band. TCE and IP samples were loaded on the same gel and processed on the same filter. Blots were vertically cropped to show appropriate expositions. Full blots are shown in the source data F4 file. (E) Co-IP showing that spastin interaction with CAND1 is stronger in HeLa Ctr-Cas9 cells compared with HIPK2-Cas9 cells. TCE from Ctr-Cas9 and HIPK2-Cas9 cells were analysed by WB or immunoprecipitated with anti-spastin Ab and analysed with indicated Abs. IgG immunoglobulins were used as IP negative control. Band intensities of co-immunoprecipitated CAND1 were normalized by band intensities of spastin immunoprecipitated and their relative values are reported as mean ± SD (n = 3). Statistical difference, unpaired t test.
Source data are available for this figure.
Source Data for Figure 4[LSA-2020-00799_SdataF4.1.xlsx][LSA-2020-00799_SdataF4.2.tif]
- Figure S3. Comparison of spastin-Flag WT and S268D levels in HeLa Ctr-Cas9 cells.
Data quantification relative to Fig 4C. Spastin-Flag intensity relative to our internal efficiency transfection control (GFP) was reported relative to M87-spastin-Flag, M1-spastin-Flag and to spastin-Flag (i.e., M1+ M87). Statistical differences, unpaired t test.
- Figure S4. MLN4924 prevents spastin polyubiquitination stabilizing its protein levels.
(A) HeLa cells were treated with 25 μg/ml cycloeximide and 0.1 μM MLN4924 or its solvent DMSO and analysed by Western blot (WB) at indicated times after treatment. Representative WB of four different experiments is reported. Cullin deneddylation is shown as MNL4924 positive control and vinculin is shown as loading control. The levels of spastin bands relative to those of vinculin were measured at each time point and reported as mean ± SEM of four different independent experiments in the right panel. Statistical differences were calculated and reported for each time point, unpaired t test. (B) HeLa cells were transfected with vectors expressing Ub-HA and pretreated 6 h posttransfection with 0.1 μM MLN4924 or its solvent, DMSO, for 16 h and treated 24 h posttransfection with 20 μM MG132 or its solvent DMSO for additional 8 h. As in Fig 2C, total cell extracts were immunoprecipitated with anti-spastin mouse monocolonal Ab, sp3G11/1. Total cell extracts and IPs were analysed by WB with anti-HA and anti-spastin Abs. Representative WB of three independent experiment is shown. The arrow indicates the position of unmodified spastin protein and the asterisk indicates a non-specific band. Cullin deneddylation is shown as MNL4924 positive control; Ponceau staining is shown as further loading control beside vinculin immunostaining. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin bands in IP and reported relative to the DMSO-treated sample as mean ± SD (n = 3). Statistical differences, unpaired t test. (C) NSC34 cells were transfected as in Fig 5A, incubated in differentiation medium and analysed by IF with anti-acetylated-tubulin Ab (green) and mitotracker (red) 5 d after siRNA transfection and differentiation for neurite swelling evaluation. Representative fields of indicated cells are reported. The arrowheads indicate neurite swelling. Scale bar, 10 μM.
- Figure 5. Restoration of spastin protein levels and neurite swelling in SPG4-(HSP) Hereditary Spastic Paraplegia models.
(A, B) NSC34 cells were transfected with 40 nM spastin-specific (siSpastin) or negative control stealth siRNAs and incubated in differentiation medium. 48 h post siRNA transfection cells were transfected with empty or HIPK2 expressing vectors and analysed by Western blot (WB) and by IF using anti-acetylated-tubulin Ab (green) and mitotracker (red) 5 d after siRNA transfection and differentiation. (A) Representative WB is shown in (A); the intensity of the spastin bands were quantified, normalized and reported relative to empty vector in siCtr and in siSpastin cells as mean ± SD of three independent experiments. Statistical differences, unpaired t test. (B) The percentage of swelling is reported in (B) as mean ± SD of three different experiments in which >100 cells were analysed. Statistical differences, ANOVA test. Representative image of neurite swelling is shown. Scale bar, 10 μM. (A, C, D) NSC34 cells were transfected as in (A) and incubated in differentiation medium. 72 h post siRNA transfection cells were treated with MLN4924 0.1 μm or its solvent DMSO and analysed 16 h after treatment. (C) Representative WB is shown in (C). Cullin deneddylation is shown as MNL4924 positive control. The intensity of the spastin bands were quantified, normalized and reported relative to solvent-treated cells in siCtr and in siSpastin as mean ± SD of three independent experiments. Statistical differences, unpaired t test. (D) The percentage of swelling is reported in (D) as mean ± SD of three different experiments in which >100 cells were analysed. Statistical differences, Anova test. Representative image of neurite swelling is shown. Scale bar, 10 μM. No dead cells were observed after treatment. (E) Representative WB showing the increase of endogenous spastin protein levels 72 h after 0.1 μM MLN4924 treatment in indicated primary cortical neurons derived from SPG4 WT (+/+) and heterozygous (+/Δ7) E5 mice. The intensity of the spastin bands were quantified, normalized and reported relative to solvent-treated cells in neurons derived from SPG4 WT (+/+) and heterozygous (+/Δ7) as mean ± SD of two independent experiments. (F) Representative WB showing the increase of endogenous spastin protein levels 16 h after 0.1 μM MLN4924 treatment in SPG4-HSP lymphoblastoid cells. The intensity of the spastin bands were quantified, normalized and reported relative to solvent-treatment in healthy donors lymphoblastoid cells and in patient’s derived SPG4-HSP lymphoblastoid cells. Statistical differences, unpaired t test.
Source data are available for this figure.
Source Data for Figure 5[LSA-2020-00799_SdataF5.1.xlsx][LSA-2020-00799_SdataF5.2.tif]
- Figure 6. Schematic model depicting the regulation of spastin protein levels.
Because S268-phosphorylation (indicated with a blue P) prevents polyubiquitination of spastin, its protein levels are the consequence of a balance between phosphorylation and degradation events depending on HIPK2 kinase activity and the function of a still unknown CRL complex. Based on this model, low HIPK2 activity reduces the spastin phosphorylated forms leading to an increase of spastin polyubiquitination with a consequent decrease of spastin protein levels due to proteasomal degradation. In contrast, high HIPK2 activity protects spastin from polyubiquitination, increasing its protein levels. When polyubiquitination is prevented using the neddylation inhibitor, MLN4924, spastin protein levels increase independently of its phosphorylation status.
Supplementary Materials
Supplemental Data 1.
[LSA-2020-00799_Supplemental_Data_1.xlsx]Identification of proteins contained in spastin-S268A and spastin-S268D immunoprecipitates from HeLa cells. The original list of proteins was filtered to include only proteins that are absent from the negative control experiments.
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